Journal of Forensic Pathology

A quick SARS-CoV-2 detection method using SmartAmp is available

^*Correspondence: Nisha Singh, Editorial Office, Journal of Forensic Pathology, UK, Email:

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Introduction

The first outbreak of the coronavirus illness 2019 (COVID-19), which is caused by the SARS-CoV-2 virus, was noted in Wuhan, China, in December 2019. Since then, it has spread quickly over the world, killing more than 4.5 million people as of September 2021, and the death toll is still rising. In January 2021, Heinrich and colleagues reported that samples taken from the nasopharynx of SARS-CoV-2-infected dead bodies could detect subgenomic RNA of the virus, indicating viral replication, and a viral culture was still possible even 35.8 hours after death, suggesting a risk of infection. Determining the cause of death more accurately and lowering the risk of infection for all parties engaged in the autopsy are therefore both dependent on the identification of SARS-CoV-2 during autopsies where the cause of death is uncertain.The quantitative real-time polymerase chain reaction (qRT-PCR) technique is the most reliable SARS-CoV-2 diagnostic method. Despite having excellent sensitivity and specificity, it need for somewhat specialised facilities and operators with technical training. The lengthy testing times (about 2-3 h) and potential for contamination associated with qRT-PCR are caused by a number of challenging procedures, including RNA extraction and reverse transcriptase reactions. Consequently, it is challenging to use qRT-PCR for on-site testing, such as autopsies or postmortem investigations after a crisis. Isothermal nucleic acid amplification has become more popular as a new technique for detecting nucleic acids in recent years.The isothermal amplification method amplifies nucleic acids by reacting at a constant temperature, as opposed to the PCR method, which amplifies nucleic acids by cycling through denaturation, annealing, and elongation at various temperatures. This has a variety of benefits, including a quicker response time, the absence of specialised machinery like a heat cycler, and a straightforward operation requiring no specialised skillsAs a result, we concentrated on the Smart Amplification (SmartAmp) method, one of the isothermal nucleic acid amplification techniques, in this study.The "SmartAmpTM 2019 New Coronavirus Detection Reagent" is an RT-SmartAmp assay that can conduct both reverse transcriptase and isothermal DNA amplification operations in a single reaction tube. It was created by DNAFORM Corporation (Yokohama, Japan) and RIKEN (Yokohama, Japan). The RNA extraction process takes about 15 minutes to complete for eight samples when combined with the specialised tools "LifeCase Smart" and "LifeCase Amp," the nucleic acid extraction reagent "Smart Extract" and these tools. Additionally, when the viral load is high, the reaction time is roughly 15 minutes, and when a sample is negative, it is roughly 40 minutes. The ability to cut the test time to less than an hour is SmartAmp's greatest benefit. Additionally, it is simple to transport because the necessary equipment is conveniently stored in three attaché cases. This makes it possible to conduct quick examinations in settings where dead bodies are handled, such as autopsies, exterior examinations, and routine mortuary handling. This will avoid infection in those who touch and work with dead corpses. The SmartAmp approach has only been evaluated and reported on as a useful SARS-CoV-2 detection method using clinical specimens; there have been no reports utilising postmortem materials. In this work, we used swabs from dead people to conduct tests using the SmartAmp technique and the qRT-PCR assay, and we compared the operation times and the acquired findings to assess the efficacy of the SmartAmp SARS-CoV-2 assay in postmortem specimens. From January to May 2021, swabs were taken from the nasopharynx, oropharynx, and anus of 11 cadavers who had undergone autopsies or postmortem biopsies at the forensic medicine departments of three Japanese universities (qRT-PCR testing revealed that five cases were positive for SARS-CoV-2 and six were negative. The postmortem period lasted 2 days to 11 days in cases that were positive and 3 days-5 days in those that were negative. There were no corpses that were far along in the process of decay. The TaqManTM 2019- nCoV Assay Kit v1, TaqManTM Fast Virus 1-Step Master Mix, and TaqManTM 2019-nCoV Control Kit v1 (all from Thermo Fisher Scientific, Franklin, MA, USA) were the testing supplies utilised for qRT-PCR. A 25-L reaction mix including 6.25 L of Master Mix, 1.25 L of primers (to the ORF1ab, N-protein, and S-protein sections, respectively), 1.25 L of internal standard primer, and 5 L of the sample was prepared in accordance with the procedure. qRT-PCR was performed using a StepOnePlus™ Real-Time PCR System. In the qRT-PCR, positive results were defined as the amplification of at least two ORF1ab, N-protein, or S-protein regions in at least one of the swabs obtained from the three sites. The identical materials and N-region primers with the same formulation, temperature, and cycling conditions were used for copy number quantification. The positive control provided by the National Institute of Infectious Diseases was diluted seven times in succession to create the calibration curve. Both the calibration curve and the samples were measured at two of these dilutions. The TaqManTM 2019- nCoV Assay Kit v1, TaqManTM Fast Virus 1-Step Master Mix, and TaqManTM 2019-nCoV Control Kit v1 (all from Thermo Fisher Scientific, Franklin, MA, USA) were the testing supplies utilised for qRT-PCR. A 25-L reaction mix including 6.25 L of Master Mix, 1.25 L of primers (to the ORF1ab, N-protein, and S-protein sections, respectively), 1.25 L of internal standard primer, and 5 L of the sample was prepared in accordance with the procedure. A StepOnePlusTM Real-Time PCR System was used for the qRTPCR procedure. 45 cycles at 50°C for 5 minutes, 95°C for 20 seconds, 95°C for 3 seconds, and 60°C for 30 seconds. In the qRT-PCR, positive results were defined as the amplification of at least two ORF1ab, N-protein, or Sprotein regions in at least one of the swabs obtained from the three sites. The identical materials and N-region primers with the same formulation, temperature, and cycling conditions were used for copy number quantification. The positive control provided by the National Institute of Infectious Diseases was diluted seven times in succession to create the calibration curve.At two of these dilutions, the calibration curve and the samples were both measured. The genotyping of single nucleotide polymorphisms and the detection of pathogens are two applications for which the SmartAmp approach has been developed. Additionally, it is utilised in clinical settings as a quick diagnostic for SARS-CoV-2.In this investigation, we extracted and amplified RNA from samples using Smart Extract and the SmartAmpTM 2019 SARS-CoV-2 detection reagents. Aspiration and straightforward centrifugation are also methods that Smart Extract is capable of using to extract RNA. The aspiration approach is usually the primary option, however the straightforward centrifugation method can be employed for extraction in highly viscous samples that are challenging to aspirate. However, the bulky and heavy hoover equipment needed for the aspiration process makes it challenging to move. In contrast, a desktop centrifuge can be used to carry out the straightforward centrifugation process. Both techniques were looked at in this study because, in contrast to clinical samples, samples taken from deceased corpses may contain skin fragments, mucous, or other pollutants that make aspiration challenging. The straightforward centrifugation procedure consistently produced false negative results among the positive samples. We decreased the volume of the extraction solution to reduce contaminants in the event that they could not be completely eliminated and PCR inhibitors persisted, and the findings were as expected. This discovery revealed that certain PCR-inhibiting contaminants were present in the postmortem specimens but were not removed by the straightforward centrifugation procedure, leading to erroneous negative results. The tabletop centrifuge that comes with this package has a weak centrifugal force (maximum 2000 g), therefore even though we were unable to locate this impurity, it is still conceivable that one may be left. The results of postmortem modifications could be one of the causes of insufficient centrifugation. A further study will focus on identifying contaminants and looking into what causes insufficient centrifugation. However, postmortem specimens can be examined using a modified basic centrifugation procedure because accurate results can be obtained by lowering the amount of RNA supplied. One instance (Case 4) had anal swabs that tested positive by qRT-PCR but negative by the SmartAmp technique. The anal swab from Case 4 had a 1.31 102 RNA copy number as assessed by qRT-PCR, which denotes a low viral load. The TaqManTM 2019-nCoV Assay Kit v1 may detect as low as 10 copies of synthetic viruses, although the SmartAmp technique, which is marginally less sensitive than qRT-PCR, can identify as many as 50 copies. Furthermore, because they contain PCR inhibitors and other chemicals, extracts from real samples, particularly those from cadavers, are predicted to have even lower sensitivity. Since the Case 4 anal swab exhibited a low viral load, it is assumed that the SmartAmp technique was unable to identify it. However, both methods produced identical results in the often tested nasopharynx and oropharynx specimens, indicating that the SmartAmp method is effective when evaluating dead bodies. Furthermore, the sample from the drowned cadaver, with an estimated postmortem interval of 7 days–11 days, yielded positive results using both the SmartAmp method and the qRT-PCR method.